Performance of the 4Kscore Test in Plasma and Serum and Stability of the Component Analytes in Clinical Samples

Background The 4Kscore Test determines a personalized risk score for aggressive prostate cancer by combining the blood sample measurements of total prostate-specific antigen (tPSA), free PSA (fPSA), intact PSA (iPSA), and human kallikrein-related peptidase 2 (hK2) with patient clinical information to generate the patient risk's score; thus, accuracy and precision of the 4Kscore depend on the reliability of these measurements. Although tPSA and fPSA are measured on a Food and Drug Administration (FDA)-approved platform, the performance of the iPSA and hK2 assays in the clinical setting has not previously been reported. Methods Analytical performance was determined for the iPSA and hK2 assays in both serum and EDTA plasma, according to Clinical and Laboratory Standards Institute guidelines. Equivalence of the 4Kscore in both sample matrices was demonstrated in a 353-patient clinical cohort, and the stability of endogenous iPSA and hK2 for at least 3 days was demonstrated in a smaller subset. Results Intralaboratory and interlaboratory precision of the iPSA and hK2 assays in both matrices was comparable with that of FDA-approved tPSA and fPSA assays (<18% for iPSA; <8% for hK2). The picogram per milliliter sensitivity and wide dynamic range of the iPSA and hK2 assays allowed for accurate measurements in the target population. The 4Kscore generated in either matrix up to 3 days after collection is equivalent to that measured within 24 h of collection (Passing–Bablok slope 95% CI: plasma, 0.999–1.034; serum, 0.997–1.040). Conclusions The robust performance of component assays and reliable stability of the endogenous analytes in clinical samples proven here ensures an accurate 4Kscore Test result.

Epitope Analysis of a Prostate-specific Antigen (PSA) C-Terminal-specific Monoclonal Antibody and New Aspects for the Discrepancy between Equimolar and Skewed PSA Assays

Background: Immunoassays to measure prostate-specific antigen (PSA) often give different values for the same patient samples, and the calibrators among commercial immunoassays are not interchangeable. We developed three novel assays to quantify the free and complexed forms of PSA in serum. Methods: We synthesized 46 peptides, which encompassed the entire PSA molecule, and determined the interactions between selected monoclonal antibodies (MAbs) and those peptides or the intact PSA molecule. Results: MAb PA313 did not cross-react with human glandular kallikrein (hK2), which has 78% amino acid homology to PSA. This MAb bound with KD = 40 nmol/L to the C-terminal peptide of PSA and distinguished between a synthetic peptide derived from PSA (PSA46A: NH2-C-R226KWIKDTIVANP237-COOH) that differed from one derived from hK2 (PSA46B: NH2-C-R226KWIKDTAANP237-COOH) by a single amino acid. Only the MAb combination of PA313/PA121 showed equimolar reactivity with PSA and with PSA complexed with α1-antichymotrypsin (PSA-ACT). The free form of PSA (F-PSA) was determined by MAbs PA313/FPA503, and the amount of complexed PSA (C-PSA) in PSA-ACT was determined by αACT/PA313. The total PSA (T-PSA) measured by either of the equimolar assays (PA313/PA121 or Tandem-R) was consistent with the sum of F-PSA and C-PSA. In contrast, T-PSA by a skewed assay (IMx) was higher than F-PSA + C-PSA when the ratio of F-PSA to T-PSA (F/T) was >0.15. T-PSA measured by IMx was nearly equal to F-PSA/0.55 + C-PSA. The coefficient 0.55 reflected different reactivities of the IMx assay with PSA-ACT and PSA. Conclusion: The discrepancy between the values measured by equimolar and skewed assays depends on the ratio of free to total PSA in the sample.

Purification and characterization of different molecular forms of prostate-specific antigen in human seminal fluid

We have developed a new procedure for purifying prostate-specific antigen (PSA) from human seminal fluid. The method is based on ammonium sulfate precipitation, hydrophobic interaction chromatography, gel filtration, and anion-exchange chromatography. It can be completed within 2 days with a recovery of intact PSA of 30%. By anion-exchange chromatography, five isoforms of PSA (A, B, C, D, and E) can be separated. The major form (PSA-B) consists of the intact enzyme, as shown by the occurrence of only one band of 33 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, and by amino acid sequencing, which reveals only one amino-terminal sequence corresponding to the reported amino-terminal sequence of intact PSA. The specific absorbance of 1 g/L PSA-B at 280 nm was 1.61, and 80% of the PSA-B formed a complex with alpha 1-antichymotrypsin, indicating that it is enzymatically active. Three cleaved forms of PSA with different nicking sites and low enzymatic activity were separated from intact PSA by ion-exchange chromatography. In addition, we isolated a glycosylation variant, PSA-A, which showed a higher isoelectric point (pI = 7.2) than PSA-B (pI = 6.9) but similar enzymatic activity; this form accounts for 5-10% of total PSA. After treatment with sialidase, PSA-A and B had the same isoelectric point value (pI = 7.7).